Lipid distribution is a fundamental feature of cellular membranes. The synthesis, remodeling, and trafficking pathways of lipids all contribute to how they are spatially arranged; in turn, lipid distribution directly modulates membrane structure and function. Yet lipid organization within the membranes of organelles, particularly morphologically complex ones, is poorly understood. The mitochondrion is an excellent system for addressing lipid distribution patterns in topologically diverse membranes. Mitochondria contain an outer membrane and an inner membrane, the latter of which is further compartmentalized into an inner boundary membrane and cristae membrane. Each mitochondrial subcompartment is enriched in specific proteins or protein complexes charged with performing distinct mitochondrial functions. In contrast, our current understanding of mitochondrial lipid distribution is comparatively rudimentary. Hence, there is a profound gap in our knowledge of the spatiotemporal distribution of mitochondrial lipids among membrane subcompartments, and how lipids in the vicinity of mitochondrial complexes may change during stress or under different functional states. Recently, we successfully implemented a strategy to measure phospholipid content in specific mitochondrial subdomains. Our approach exploited the well-established ability of styrene-maleic acid (SMA) copolymers to extract nanodiscs from membranes that contain proteins and their surrounding lipids, termed SMA lipoprotein particles (SMALPs). Combining our expertise in membrane biochemistry/biophysics and mitochondrial physiology with our expertise in SMALP approaches, in the present application we will take this technology to the next level, using it to analyze membrane compartment- and complex-specific phospholipids and spatiotemporal changes that may occur under biologically relevant states. In Aim 1, we will test the hypothesis that local lipid composition is tuned to distinct functions of different membrane regions. To test this hypothesis, the phospholipid composition in the immediate vicinity of protein complexes that localize to distinct mitochondrial subcompartments will be determined in affinity purified SMALPs isolated from both mitochondrial and model membranes. Results generated will fill profound knowledge gaps regarding the lipid profiles of different mitochondrial subcompartments and how the distribution of lipids around specific complexes may be regulated. In Aims 2 and 3, we will exploit the enabling SMALP- based approach to determine how acyl chain remodeling of the mitochondrial-specific phospholipid, cardiolipin, promotes mitochondrial function. Specifically, in Aim 2 we will combine genetic models with our SMALP approaches to test the hypothesis that cardiolipin remodeling is mechanistically linked to cardiolipin distribution. In Aim 3, genetic models, dietary supplementation, and oxidative stress conditions will be combined with our SMALP approaches to test the hypothesis that CL remodeling acts as a mitochondrial quality control mechanism important for handling mitochondrial oxidative stress. More broadly this work expands our understanding of basic mechanisms contributing to mitochondrial myopathies and cardiovascular disease.